WO2006046978A2

WO2006046978A2 - Cationic peptide-mediated transformation

Info

Publication number: WO2006046978A2
Application number: PCT/US2005/022705
Authority: WO
Inventors: Charles Nicolette; Irina Tcherepanova
Original assignee: Argos Therapeutics, Inc.
Priority date: 2004-06-28
Filing date: 2005-06-28
Publication date: 2006-05-04
Also published as: WO2006046978A3

Abstract

The invention provides novel methods of cationic peptide-mediated transformation of RNA into cells, cationic peptides useful in such methods, related noncovalent cationic peptide-nucleic acid complexes and cells transformed with such complexes. The invention further provides novel cationic peptides containing embedded MHC helper epitopes. Such cationic peptides are particularly useful for the transformation of antigen presenting cells, such as dendritic cells.

Description

CATIONIC PEPTIDE-MEDIATED TRANSFORMATION

CROSS-REFERENCE TO RELATED INVENTIONS

This application claims priority to U.S. provisional application 60/583,579 filed 28 June 2004, the content of which is incorporated by reference.

FIELD OF THE INVENTION

The invention relates to methods of transforming cells. More specifically, the invention relates to methods of cationic peptide-mediated transformation of RNA into cells, cationic peptides useful in such methods, related cationic peptide-nucleic acid complexes and cells transformed with such complexes.

BACKGROUND OF THE INVENTION

Traditional methods of transforming cells with nucleic acids include lipid-mediated transformation, transfection with polymeric DNA binding cations such as poly-L-lysine, dendrimers or polyethylenimine, electroporation, calcium phosphate transformation and viral- mediated transfection. However, these methods are limited due to low efficiency, cytotoxicity, constraints on upper limits on nucleic acid size, and/or adverse events in mammals.

It is now known that a variety of peptides, and in particular, cationic peptides, can translocate into cells and also mediate the translocation of proteins into cells. These cationic peptides include HIV-I Tat peptide (YGRKKRRQRRR; SEQ ID NO:1), R₉-Tat (GRRRRRRRRRPPQ; SEQ ID NO:2), the protein transduction domain (PTD) of the Drosophila Antennapedia Homeodomain, also known as AntP and penetratin (RQIKIWFQNRRMKWKK; SEQ ID N0:3), HIV-I Rev-(34-50)

(TRQARRNRRRRWRERQR; SEQ ID NO:4), FHV Coat (35-49) (RRRRNRTRRNRRRVR; SEQ ID NO: 5), Brome Mosaic Virus (BMV) Gag-(7-25) (KMTRAQRRAAARRNRWTAR; SEQ ID NO:6), HTLV-II Rex-(4-16) (TRRQRTRRQRRNR; SEQ ID NO:7), CCMV Gag-(7- 25) (KLTRAQRRAAARKNKRNTR; SEQ ID NO:8), P22 N-(14-30) NAKTRRHERRRKLAIER; SEQ ID NO:9), W/R (RRWRRWWRRWWRRWRR; SEQ ID NO:10), NLS (TPPKKKRKVEDP; SEQ ID NO:11), AIkCWK₁₈

(CWKKKKKKXKKKKKKKKKKK; SEQ ID NO: 12), DiCWK₁₈ (Ki₈WCCWKi₈; SEQ ID NO: 13), Transportan (GWTLNSAGYLLGKINLKALAALAKKIL; SEQ ID NO: 14, a chimera of the first 12 amino acids of the neuropeptide galanin fused by an e -amino of a lysine with a 14 residue mastoparan, a wasp venom peptide), KigRGD (K₁₆GGCRGDMKGCA K₁₆RGD; SEQ ID NO:15), Pl (KI₆GGCMFGCGG; SEQ ID NO: 16), P2 (KielCRRARGDNPDDRCT; SEQ ID NO: 17), P3a (VAYISRGGVSTYYSDTVKGRFTFQKYNKRA; SEQ ID NO: 18), P9.3 (IGRIDPANGKTKYAPKFQDKATRSNYYGNSPS; SEQ ID NO:19), Kplae (KieGGPLAEIDGIELGA; SEQ ID NO:20), cKplae (K_]6GGPLAEIDGIELCA; SEQ ID NO:21), MPG (GALFLGFLGGAAGSTMGAWSQPKSKRKV; SEQ ID NO:22, a chimera of a fragment of the HIV gp41 protein and the nuclear localization signal (NLS) of the SV40 large T antigen), (LAR)_n oligomer, a branching peptide which is a chimera of the NLS of the SV40 large T antigen and a lysine pentapeptide and branched arginine peptides, and a variety of other peptides (for reviews, see Schwartz et al. Current Opinion in Molecular Therapeutics (2000) 2(2), Tung and Weissleder, Adv Drug Delivery Rev (2003) 55:281-294, Vives, J MoI Recognit (2003) 16:265-271 and Futaki et al Current Protein Pept Sci (2003) 4:87-96).

Several groups have reported peptide-mediated transfection of covalently coupled DNA and RNA. For example, covalent conjugates of antisense oligodeoxynucleotides to AntP and Tat have been shown to down regulate target gene expression. (Allinquant et al. J Cell Biol (1995) 128:919-927 and Troy et al. J Neurosci (1996) 16:857-861). In addition, Schmitz et al. (2003 Meeting of the European Life Scientist Organization in Dresden, Poster Abstract #432) describes transfection of mammalian cells and down regulation of mRNA expression by siRNA covalently linked to AntP by a disulfide bond.

In addition, there have been reports of peptide-mediated transfection of non- covalently coupled nucleic acids. For example, WO 98/29541 describes the polymerization of a 12-merNLS peptide from SV40 large T antigen upon a DNA template using the cross- linker DPDPB. The NLS peptide was polymerized by a cross linking agent into a NLS multimer which encased the DNA. Transfection of the NLS multimer with an encased DNA template encoding luciferase resulted in luciferase expression. Unfortunately, this transfection method is hampered by the added step of cross-linking the peptide upon a DNA template.

Morris et a (Nuc Acids Res (1997) 25:2730-2736) disclose transfection of fibroblast cells with single and double stranded oligodeoxynucleotides (18 and 36-mers) mediated by a non-covalently complexed 27-mer MPG peptide. The MPG peptide contains a hydrophobic domain derived from HIV gp41 (GALFLGFLGAAGSTMGA; SEQ ID NO:23) linked by a short spacer peptide (WSQ) to the hydrophilic NLS of SV40 large T-antigen (PKSKRKV; SEQ ID NO: 24). Transfected labeled oligodeoxynucleotides were localized to the nucleus.

U.S. Patent Application Publication No. 20030087810 discloses transfection of mammalian cells with 18 and 20 residue antisense oligodeoxynucleotides non-covalently complexed with a peptide comprising a fusion of a hydrophobic N-terminal DNA-binding domain from the SV40 large T-antigen and a hydrophilic C-terminal nuclear localization signal (NLS) consisting of a basic stretch of five consecutive positively charged residues from the same protein. The DNA binding domain is thought to be required for efficient crossing of the cell membrane, while the NLS domain is required for nuclear targeting. Pretreatment of cells with chloroquine, a lysosomotropic agent, was necessary for antisense effect in PC-3 prostate carcinoma cells, but was not required in T24 bladder carcinoma cells.

U.S. Patent Application Publication No. 20030125242 describes transfection of several mammalian cell lines with noncovalent complexes of (PKKKRKVG)₄ (SEQ ID NO:25; four linear repeats of the nuclear localization signal (NLS) of the SV40 large T antigen) and DNA encoding luciferase. Transfected cells expressed luciferase. According to the specification, the NLS sequence was repeated 4 times in order to achieve enough positive charges for a stable electrostatic complex with DNA. Comparison of the efficiency of DNA transfection using two, three or four repeats of a different NLS, C(YGRKKRRQRRRG)_2-4 (SEQ ID NO:26), showed that 3-mers were more efficient than 2-mers and 4-mers. The '242 application suggests, but does not demonstrate, transfection with RNA, such as ribozymes.

U.S. 6,479,464 discloses the transfection of human dendritic cells with a cationic peptide/DNA complex in the presence of chloroquine. EP 0 880 360 discloses vaccine compositions comprising a nucleic acid (DNA) encoding a first epitope and a peptide comprising a second epitope, and proposes that the nucleic acid and peptide are taken up by an antigen presenting cell in the vaccinated mammal. Preferred peptides are cationic. The peptide can contain two domains, an antigenic domain and a DNA-binding domain. In vitro transfection of mature dendritic cells was demonstrated using a composition comprising a DNA plasmid encoding GFP, a cationic peptide

(KKXPKKYZBPAJKKXPKKYZBPAJKKXPKKYZBPAJC; SEQ ID NO:27, where: X is Serine, Threonine or Proline; Y is Alanine or Valine; Z is Alanine, Threonine or Proline; B is Lysine, Alanine, Threonine or Valine; and J is Alanine or Valine), an N-palmityl derivative of the cationic peptide and chloroquine. RNA-peptide compositions were not disclosed.

Methods of RNA transfection are not as well developed as DNA transfection methods. With the growing importance of technologies such as RNA loading of antigen presenting cells, it is critical to develop improved methods of RNA transfection. However, there have been few reports of successful peptide-mediated RNA transfection. One method requires covalently linking the peptide to polyethylenimine (PEI), a highly branched aliphatic polyamine. Specifically, Bettinger et al. (Nuc Acids Res (2001) 29:3883-3891) disclose transfection of mammalian cells with mRNA in the presence of a melittin peptide cross- linked to PEI-2 kDa. Melittin (CIGAVLKVLTTGLPALISWIKRKRQQ; SEQ ID NO:28) is a membrane active peptide from bee-venom that inserts into lipid membranes and promotes leakage, and at high concentrations causes membrane disruption. Accordingly, treatment of cells with the PEI-melittin complex results in poor cell viability.

EP 1 083 232 discloses the vaccination of mice in the ear pinna with non-covalent capped /3-gal-RNA-protamine (cationic peptides of about 4.2 kD from salmon sperm) complex. The (3-gal-RNA was expressed in ear pinna tissue and generated a /3-gal specific CTL response. The authors speculate, but do not demonstrate, that the RNA vaccine may transduce dendritic cells in vivo. In vitro transfection of dendritic cells with RNA:cationic peptide complexes is not suggested.

Scheel et al. (2004) Eur. J. Immunol. 34:537-547 disclose that protamine condensed mRNA induces maturation of mouse dendritic, cells, but do not demonstrate nor suggest transfection of the mouse cells.

Thus, there exists a need to develop improved methods to efficiently deliver RNA to cells, while retaining cell viability. The present invention satisfies this need and provides related advantages as well.

BRIEF SUMMARY OF THE INVENTION

Applicants have discovered that cationic peptides can mediate the efficient transfection of cells with RNA without the need for covalent bonding of the RNA and peptide, or the need for PEI-peptide complexes. Thus, the invention provides a method of transforming cells, comprising: contacting a cell with a cationic peptide and a RNA. The contacting may be performed either in vivo or in vitro. In a preferred embodiment, the cell(s) is contacted with a non-covalent complex comprising a cationic peptide and a RNA. Preferably, the non-covalent complex is formed prior to contacting the cell, by steps of (a) adding the cationic peptide to the RNA to form a mixture, and (b) incubating the mixture for a time sufficient to allow the formation of a non-covalent complex between the cationic peptide and the RNA.

The invention also provides cationic peptides useful for transfecting cells. Preferably, the peptide is 8 to 24 amino acids in length. More preferably, the peptide is 10-22 amino acids in length, most preferably 12-20 amino acids in length. In one embodiment of the invention, the cationic peptides of the invention do not contain a nuclear localization signal. Preferably, the cationic peptide consists essentially of a peptide transduction domain. In a preferred embodiment, the cationic peptide is preferentially localized to the cytoplasm of said cell.

In further embodiments of the invention, the cationic peptide is selected from the group consisting of AntP (RQIKIWFQNRRMKWKK; SEQ ID NO:3), HIV Tat (GRKKRRQRRRPPQ; SEQ ID NO:29) or HIV Tat dimer

(CGRKKRRQRRRPPQGRKKRRQRRRPPQ; SEQ ID NO:30) and a combination thereof. Preferably, the cationic peptide comprises a MHC helper epitope, such as a MHC class I helper epitope or a MHC class II helper epitope. In one embodiment, the helper epitope is embedded in the cationic peptide. Preferred embodiments of helper epitopes embedded in cationic peptides are RRKAQYIKANSKFIGITELKRH (SEQ ID NO:31), KKKHIEKYLKKIKNSKKK (SEQ ID NO:32), KKKVIKGGRHLIFCHSKKKCDKKK (SEQ ID NO:33), KKKPKYVRQNTLKLATKKK (SEQ ID NO:34) and KKKKALENKKKQLGAGGKNKKK (SEQ ID NO:35).

The cationic peptides are useful for mediating transfection of any RNA into any type of cell. Preferred RNAs are mRNA, antisense RNA, RNAj, amplified RNA and ribozymes. Preferably, the RNA is a translatable RNA. In one embodiment, the RNA is isolated from one or more tumor (neoplastic) cells, such as cancer cells. In another embodiment, the RNA is isolated from one or more pathogens, such as HIV or HCV.

The invention further provides compositions comprising noncovalent cationic peptide-RNA complexes and cells transfected with cationic peptide-RNA complexes. Preferred cells for transfection are eukaryotic cells. The cationic peptide-RNA complexes of the invention are particularly useful for transforming antigen presenting cells of the immune system, such as immature and mature dendritic cells.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

As used in the specification and claims, the singular form "a," "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a cell" includes a plurality of cells, including mixtures thereof.

All numerical designations, e.g., pH, temperature, time, concentration, and molecular weight, including ranges, are approximations which are varied ( + ) or ( - ) by increments of 0.1. It is to be understood, although not always explicitly stated that all numerical designations are preceded by the term "about". It also is to be understood, although not always explicitly stated, that the reagents described herein are merely exemplary and that equivalents of such are known in the art.

The term "antigen" is well understood in the art and includes substances which are immunogenic, i.e., immunogen. It should also be understood will be appreciated that the use of any antigen is envisioned for use in the present invention and thus includes, but is not limited to a self-antigen (whether normal or disease-related), an infectious antigen (e.g., a microbial antigen, viral antigen, etc.), or some other foreign antigen (e.g., a food component, pollen, etc.). The term "antigen" or alternatively, "immunogen" applies to collections of more than one immunogen, so that immune responses to multiple immunogens may be modulated simultaneously. Moreover, the term includes any of a variety of different formulations of immunogen or antigen.

A "native" or "natural" or "wild-type" antigen is a polypeptide, protein or a fragment which contains an epitope, which has been isolated from a natural biological source, and which can specifically bind to an antigen receptor, when presented as an MHC/peptide complex, in particular a T cell antigen receptor (TCR), in a subject.

The term "antigen presenting cells (APC)" refers to a class of cells capable of presenting one or more antigens in the form of antigen-MHC complex recognizable by specific effector cells of the immune system, and thereby inducing an effective cellular immune response against the antigen or antigens being presented. While many types of cells may be capable of presenting antigens on their cell surface for T-cell recognition, only dendritic cells (professional APCs) have the capacity to present antigens in an efficient amount and further to activate naive T-cells for cytotoxic T-lymphocyte (CTL) responses. APCs include, but are not limited to, macrophages, B-cells and dendritic cells, such as immature dendritic cells, mature dendritic cells and Langerhans cells.

By "tumor" or "neoplasm" is meant a mass of abnormal tissue which may resemble normal tissues in structure, typically performs no useful function, and which grows at the expense of the body. Tumors can benign or malignant. The term "cancer" is generally refers to a malignant tumor, and is typically characterized by an abnormal presence of cells which exhibit relatively autonomous growth, so that a cancer cell exhibits an aberrant growth phenotype characterized by a significant loss of cell proliferation control. In various embodiments, the tumor affects cells of the bladder, blood, brain, breast, colon, digestive tract, lung, ovaries, pancreas, prostate gland, or skin. The definition of a tumor cell, as used herein, includes not only a primary tumor cell, but also any cell derived from a tumor cell ancestor. This includes metastasized cancer cells, and in vitro cultures and cell lines derived from tumor cells. Tumors includes but are not limited to, solid tumors, liquid tumors, hematologic malignancies, renal cell cancer, melanoma, breast cancer, prostate cancer, testicular cancer, bladder cancer, ovarian cancer, cervical cancer, stomach cancer, esophageal cancer, pancreatic cancer, lung cancer, neuroblastoma, glioblastoma, retinoblastoma, leukemias, myelomas, lymphomas, hepatoma, adenomas, sarcomas, carcinomas, blastemas, etc. Preferred tumors for treatment using the compositions and methods of the invention are renal cell carcinoma, melanoma and chronic lymphocytic leukemia. When referring to a type of cancer that normally manifests as a solid tumor, a "clinically detectable" tumor is one that is detectable on the basis of tumor mass; e.g., by such procedures as CAT scan, magnetic resonance imaging (MRI), X-ray, ultrasound or palpation. Biochemical or immunologic findings alone may be insufficient to meet this definition.

For the purposes of the invention, "cationic peptide" means a peptide that is positively charged at pH 7. The positive charge arises from amino acid residues with basic side chains. Positively charged amino acids include lysine (K), arginine (R), histidine (H) and amino acid analogs thereof.

"Cell" and "target cell" are intended to include any individual cell, multiple cells, cell culture or cell in an organism that can be or have been recipients for transfection with the cationic peptide-RNA complexes of the invention. It also is intended to include progeny of a single cell, and the progeny may not necessarily be completely identical (in morphology or in genomic or total DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation. The cells may be prokaryotic or eukaryotic, in vivo or in vitro, and include, but are not limited to, bacterial cells, yeast cells, animal cells, and mammalian cells, including human cells. Preferably the target cell is an antigen presenting cell, most preferably a dendritic cell.

As used herein, the term "consisting essentially of shall mean excluding other elements of any essential significance to the combination. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method, biological buffers and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives, and the like. For example, a peptide consisting essentially of SEQ ID NO:A will not contain more than three amino acid residues on each of the amino and carboxy terminus of SEQ ID NO: A. Also, a composition consisting essentially of a cationic peptide and a RNA will not be covalently linked to a transfection agent, such as PEL

The term "dendritic cells" (DCs) refers to a diverse population of morphologically similar cell types found in a variety of lymphoid and non-lymphoid tissues, Steinman (1991) Ann. Rev. Immunol. 9:271-296. Dendritic cells constitute the most potent and preferred APCs in the organism. While the dendritic cells can be differentiated from monocytes, they possess distinct phenotypes. For example, a particular differentiating marker, CD 14 antigen, is not found in dendritic cells but is possessed by monocytes. Also, mature dendritic cells are not phagocytic, whereas the monocytes are strongly phagocytosing cells. It has been shown that mature DCs can provide all the signals necessary for T cell activation and proliferation.

As used herein, "expression" refers to the processes by which polynucleotides are transcribed into mRNA and mRNA is translated into peptides, polypeptides, or proteins. If the polynucleotide is derived from genomic DNA of an appropriate eukaryotic host expression may include splicing of the mRNA. Regulatory elements required for expression include promoter sequences to bind RNA polymerase and transcription initiation sequences for ribosome binding. For example, a bacterial expression vector includes a promoter such as the lac promoter and for transcription initiation the Shine-Dalgarno sequence and the start codon AUG (Sambrook et al. (1989) supra). Similarly, a eukaryotic expression vector includes a heterologous or homologous promoter for RNA polymerase II, a downstream polyadenylation signal, the start codon AUG, and a termination codon for detachment of the ribosome. Such vectors can be obtained commercially or assembled by the sequences described in methods known in the art, for example, the methods herein below for constructing vectors in general.

As used herein, the term "inducing an immune response in a subject" is a term understood in the art and intends an increase of at least about 2-fold, or alternatively at least about 5-fold, or alternatively at least about 10-fold, or alternatively at least about 100-fold, or alternatively at least about 500-fold, or alternatively at least about 1000-fold or more in an immune response to an antigen (or epitope) which can be detected or measured, after introducing the antigen (or epitope) into the subject, relative to the immune response (if any) before introduction of the antigen (or epitope) into the subject. An immune response to an antigen (or epitope), includes but is not limited to, production of an antigen-specific (or epitope-specific) antibody, and production of an immune cell expressing on its surface a molecule which specifically binds to an antigen (or epitope). Methods of determining whether an immune response to a given antigen (or epitope) has been induced are well known in the art. For example, antigen-specific antibody can be detected using any of a variety of immunoassays known in the art, including, but not limited to, ELISA, wherein, for example, binding of an antibody in a sample to an immobilized antigen (or epitope) is detected with a detectably-labeled second antibody (e.g., enzyme-labeled mouse anti-human Ig antibody).

The term "isolated" means separated from constituents, cellular and otherwise, in which the RNA, peptide, polypeptide, protein, cell, etc., are normally associated with in nature. For example, with respect to a naturally occurring peptide, an isolated peptide is one that is separated from the cellular components it is normally associated with. With respect to peptides that are fragments of a naturally occurring protein, an isolated peptide in separated from the amino and carboxy sequences with which it is normally associated. With respect to a polynucleotide, an isolated polynucleotide is one that is separated from the 5' and 3' sequences with which it is normally associated in the chromosome. As is apparent to those of skill in the art, a non-naturally occurring polynucleotide, peptide, polypeptide, protein, or fragments thereof, does not require "isolation" to distinguish it from its naturally occurring counterpart. In addition, a "concentrated", "separated" or "diluted" polynucleotide, peptide, polypeptide, protein, antibody, or fragment(s) thereof, is distinguishable from its naturally occurring counterpart in that the concentration or number of molecules per volume is greater than "concentrated" or less than "separated" than that of its naturally occurring counterpart. A polynucleotide, peptide, polypeptide, protein, antibody, or fragment(s) thereof, which differs from the naturally occurring counterpart in its primary sequence or for example, by its glycosylation pattern, need not be present in its isolated form since it is distinguishable from its naturally occurring counterpart by its primary sequence, or alternatively, by another characteristic such as its glycosylation pattern. Although not explicitly stated for each of the inventions disclosed herein, it is to be understood that all of the above embodiments for each of the compositions disclosed below and under the appropriate conditions, are provided by this invention. Thus, a non-naturally occurring polynucleotide is provided as a separate embodiment from the isolated naturally occurring polynucleotide. A protein produced in a bacterial cell is provided as a separate embodiment from the naturally occurring protein isolated from a eukaryotic cell in which it is produced in nature.

For the purposes of the invention, "noncovalently complexed" refers to a noncovalent chemical bond or cohesion between a peptide and nucleic acid of the invention in which, in contrast to a covalent bond, no electrons are shared between the peptide and the nucleic acid. Preferably, the noncovalent complex is formed through ionic bonding of the positively charged cationic peptide and negatively charged polynucleotide. The cationic peptides of the invention are not covalently linked to PEL

The terms "nuclear localization sequence" or "nuclear localization signal" (NLS) are used interchangeably and refer to an amino acid sequence which induces transport of itself or of a complex containing it into the nucleus of a cell. For the purposes of the invention, a nuclear localization signal is defined as an amino acid sequence which causes the peptide to predominately target or localize in the nucleus of a cell. Methods for measuring and detecting nuclear translocation of peptides are known to those skilled in the art. See, for example, U.S. Patent Application Publication No: US 2003/0125242, the contents of which are incorporated by reference. An example of a nuclear localization signal is the NLS of SV40 large T antigen, which has the sequence PKKKRKVG (SEQ ID NO:36).

"Pathogen", as used herein, refers to any disease causing organism or virus, and also to attenuated derivatives thereof. Such pathogens include, but are not limited to, bacterial, protozoan and fungal pathogens such as Helicobacter, such as Helicobacter pylori, Salmonella, Shigella, Enterobacter, Campylobacter, various mycobacteria, such as Mycobacterium leprae, Bacillus anthracis, Yersinia pestis, Francisella tularensis, Brucella species, Leptospira interrogans, Staphyloccus, such as S. aureus, Streptococcus, Clostridum, Candida albicans, Plasmodium, Leishmania, Trypanosoma, and viral pathogens such as human immunodeficiency virus (HIV), Hepatitis C Virus (HCV), HPV, CMV, HTLV, herpesvirus (e.g., herpes simplex virus type 1, herpes simplex virus type 2, coronavirus, varicella-zoster virus, and Epstein-Barr virus), papilloma virus, influenza virus, hepatitis B virus, poliomyelitis virus, measles virus, mumps virus, and rubella virus. Preferred pathogens are HIV and HCV.

For the purposes of the invention, "peptide" refers to a compound of 8 to 40, preferably 9 to 30, more preferably 10-24, most preferably 12-20 subunit amino acids, amino acid analogs, peptidomimetics, or a combination thereof. The subunits may be linked by peptide bonds. In another embodiment, the subunit may be linked by other bonds, e.g. ester, ether, etc. As used herein the term "amino acid" refers to either natural and/or unnatural or synthetic amino acids, including glycine and both the D or L optical isomers, and amino acid analogs, non-classical amino acids, peptidomimetics and various "designer" amino acids (e.g., /3-methyl amino acids, C-ce-methyl amino acids, and N-α-methyl amino acids, etc.) and mixes thereof.

A "pharmaceutical composition" is intended to include the combination of an active agent with a carrier, inert or active, making the composition suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.

As used herein, the term "pharmaceutically acceptable carrier" encompasses any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, and emulsions, such as an oil/water or water/oil emulsion, and various types of wetting agents. The compositions also can include stabilizers and preservatives. For examples of carriers, stabilizers and adjuvants, see Martin REMINGTON'S PHARM. SCL, 18th Ed. (Mack Publ. Co., Easton (1990)).

An "effective amount" is an amount sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations, applications or dosages.

"Polyethylenimine" ("PEI") refers to a highly branched aliphatic polyamine characterized by the repeating chemical unit denoted as -(CH₂-CH₂-NH)-. The most prominent feature of PEI is its extremely high cationic charge density.

The term "RNA" refers to polymeric forms of ribonucleotides of any length, wherein the ribonucleotides or ribonucleotide analogs are joined together by phosphodiester bonds. The term "RNA" includes, for example, single-stranded, double-stranded and triple helical molecules, primary transcripts, mRNA, tRNA, rRNA, ribozymes, in vitro transcripts, in vitro synthesized RNA, branched polyribonucleotides, isolated RNA of any sequence, RNA; (e.g., dsRNA; and siRNAj) and the like. A RNA molecule may also comprise modified ribonucleotides. Preferably, the RNA is a translatable RNA (mRNA). Translatable RNA or mRNA will contain a ribosome binding site and start codon. Preferably, the mRNA will also contain a 5' cap, stop codon and polyA tail.

The term targeting, as used herein, means that upon introduction into a cell, the cationic peptide or cationic peptide-RNA complex will preferentially localize in a certain compartment of the cell, such as the cytoplasm or endoplasmic reticulum, nucleus, mitochondria, etc.

The invention provides novel methods of cationic peptide-mediated transfection of cells with RNA. The peptides of the invention can mediate transformation of RNA into any cell. In contrast to the prior art, the methods of the invention do not require covalent bonding of the RNA and peptide, nor covalent coupling of the peptide to polyethylenimine (PEI). Accordingly, the nucleic acid is more likely to retain its biological function once inside the cell, and the need for the extra step of covalently bonding step is eliminated. Moreover, in comparison to prior art transformation methods, such as electroporation, cationic lipids or dendrimers, the transformation methods of the invention result in greater cell viability and transformation efficiency.

Thus, the invention provides a method of transforming a cell, comprising: contacting a cell with a complex comprising a cationic peptide and a RNA, wherein said peptide and said RNA are noncovalently complexed. As used herein, transforming and transfecting are used interchangeably and refer to the introduction of a RNA into a cell.

Cationic peptides of the invention

The cationic peptides on the invention are preferably 8 to 40 amino acids in length, more preferably 9 to 30 amino acids in length, even more preferably 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 amino acids in length, and most preferably 10-20 amino acids in length. Peptides conjugated to polyethylenime are specifically excluded from the cationic peptides of the invention.

The cationic peptides can be isolated from a natural source, such as a cell or tissue, but in preferred embodiments, the cationic peptides are manufactured synthetically or by recombinant nucleic acid technology. Such methods are well known to those of skill in the art. For the purposes of the invention, cationic peptides are not used as an unpurified extract from a cell or tissue. The cationic peptides of the invention have a net positive charge at pH 7 due to their content of basic (cationic) amino acids, but may also include uncharged and negatively charged amino acid residues. Amino acids with basic side chains are positively charged (cationic) at pH 7, and include lysine (K), arginine (R) and histidine (H). Preferred basic amino acid residues for use in the cationic peptides of the invention are lysine and arginine.

Amino acids with acidic side chains are negatively charged (anionic) at pH 7 include aspartic acid (D) and glutamic acid (E). Amino acids with uncharged polar side chains at pH 7 include asparagine (N), glutamine (Q), serine (S), threonine (T) and tyrosine (Y). Amino acids with nonpolar side chains include alanine, (A), leucine (L), proline (P), methionine (M), glycine (G), valine (V), isoleucine (I), phenylalanine (F), tryptophan (W) and cysteine (C).

Amino acids that confer useful chemical and/or structural properties can be used in the peptides of the invention. For example, peptides comprising D-amino acids will be resistant to L-amino acid-specific proteases found in vivo. In another embodiment, a peptide may be generated that incorporates a reduced peptide bond, i.e., Ri — CH₂NH — R₂ where R₁, and R₂ are amino acid residues or sequences. A reduced peptide bond may be introduced as a dipeptide subunit. Cationic peptides containing reduced peptide bonds would be resistant to peptide bond hydrolysis, e.g., protease activity and metabolic breakdown, and therefore would have extended half-lives.

In addition to the twenty naturally-occurring amino acids and their homoanalogs and noranalogs, several other classes of alpha amino acids can be employed in the present invention. Examples of these other classes include D-amino acids, N^-alkyl amino acids, alpha-alkyl amino acids, cyclic amino acids, chimeric amino acids, and miscellaneous amino acids. These non-natural amino acids have been widely used to modify bioactive polypeptides to enhance resistance to proteolytic degradation and/or to impart conformational constraints to improve biological activity (Hruby et al. (1990) Biochem. J. 268:249-262; Hruby and Bonner (1995) Methods in Molecular Biology 35:201-240).

The most common N^α-alkyl amino acids are the N^α-methyl amino acids, such as N⁰¹- methyl cysteine (nK), N^α-methyl glycine (nG), N^α-methyl leucine (nL), N^-methyl lysine (nK), and N^α-methyl valine (nV). Examples of α-alkyl amino acids include α-methyl alanine (mA), α-aminoisobutyric acid (aiB), α-methyl proline (mP), α-methyl leucine (mL), α-methyl valine (mV), ce-methyl-alpha-aminobutyric acid (ty), diethylglycine (deG), diphenylglycine (dpG), and dicyclohexyl glycine (dcG) (Balaram (1992) Pure & Appl. Chem. 64:1061-1066; Toniolo et al. (1993) Biopolymers 33:1061-1072; Hinds et al. (1991) Med. Chem. 34:1777- 1789).

Examples of cyclic amino acids include 1 -amino- 1 -cyclopropane carboxylic acid (cG), 1 -amino- 1-cyclopentane carboxylic acid (Ac5c), 1 -amino- 1-cyclohexane carboxylic acid (Ac6c), aminoindane carboxylic acid (ind), tetrahydroisoquinoline carboxylic acid (Tic), and pipecolinic acid (Pip) (C. Toniolo (1990) Int'l. J. Peptide Protein Res. 35:287-300; Burgess et al. (1995) J. Am. Chem. Soc. 117:3808-3819). Examples of chimeric amino acids include penicillamine (Pe), combinations of cysteine with valine, 4R- and 4S- mercaptoprolines (Mpt), combinations of homocysteine and proline and 4R- and 4S- hydroxyprolines (hyP) and a combination of homoserine and proline. Examples of miscellaneous alpha amino acids include basic amino acid analogs such as ornithine (Or), N^e- methyl lysine (mK), 4-pyridyl alanine(pyA), 4-piperidino alanine (piA), and 4- aminophenylalanine; acidic amino acid analogs such as citrulline (Cit), and 3-hydroxyvaline; aromatic amino acid analogs such as 1-naphthylalanine (1-Nal), 2-naphthylalanine (2-Nal), phenylglycine (pG), 3,3-diphenylalanine (dpA), 3-(2-thienyl)alanine (Thi), and halophenylalanines (e.g., 2-fluorophenylalanine and 4-chlorophenylalanine); hydrophobic amino acid analogs such as t-butylglycine (i.e., tertiary leucine (tL)), 2-aminobutyric acid (Abu), cyclohexylalanine (Cy), 4-tetrahydropyranyl alanine (tpA), 3,3-dicyclohexyl alanine (dcA), and 3,4-dehydroproline.

In addition to alpha-amino acids, others such as beta amino acids can also be used in the present invention. Examples of these other amino acids include 2-aminobenzoic acid (Abz), /3-aminopropanoic acid (j8-Apr), γ-aminobutyric acid (γ-Abu), and 6-aminohexanoic acid (-Ahx). Carboxylic acids such as 4-chlorobutyric acid (By) and 3-chloropropionic acid (Pp) have also been used as the first residue on the N-terminal in the synthesis of cyclic thioether peptides.

In addition, peptidomimetics and peptidomimetic bonds, such as ester bonds, are useful in cationic peptides of the invention. Amino acid analogs and peptidomimetics include, but are not limited to: LL-Acp (LL-3-amino-2-propenidone-6-carboxylic acid), a /3-turn inducing dipeptide analog (Kemp et al., 1985, J. Org. Chem. 50:5834-5838); /3-sheet inducing analogs (Kemp et al., 1988, Tetrahedron Lett. 29:5081-5082); /3-turn inducing analogs (Kemp et al, 1988, Tetrahedron Lett. 29:5057-5060); α-helix inducing analogs (Kemp et al, 1988, Tetrahedron Lett. 29:4935-4938); 7-turn inducing analogs (Kemp et al, 1989, J. Org. Chem. 54: 109-115); and analogs provided by the following references: Nagai and Sato, 1985, Tetrahedron Lett. 26:647-650; DiMaio et al, 1989, J. Chem. Soc. Perkin Trans. P. 1687; also a GIy- Ala turn analog (Kahn et al., 1989, Tetrahedron Lett. 30:2317); amide bond isostere (Jones et al., 1988, Tetrahedron Lett. 29:3853-3856); tetrazole (Zabrocki et al., 1988, J. Am. Chem. Soc. 110:5875-5880); DTC (Samanen et al., 1990, Int. J. Protein Pep. Res. 35:501- 509); and analogs taught in Olson et al., 1990, J. Am. Chem. Sci. 112:323-333 and Garvey et al., 1990, J. Org. Chem. 56:436. Conformationally restricted mimetics of beta turns and beta bulges, and peptides containing them, are described in U.S. Pat. No. 5,440,013.

Numerous cationic peptides having the ability to translocate into cells are known to those skilled in the art and are useful in the methods, cationic peptide-RNA complexes and transformed cells of the invention. These include, but are not limited to, polyarginine, e.g., 7 to 15 arginines, preferably 8, 9, 10, 11 or 12 arginines (see Matsui et al. Current Protein and Peptide Science (2003) 4:151-157), polylysine, the protein transduction domain (PTD) of the HIV-I Tat protein and fragments thereof, such as

Ta^_7-57 (YGRKKRRQRRR; SEQ ID NO: 1), Tat variants such as YARRRRRRRRR (SEQ ID NO:37), optimized Tat peptides such as YARKARRQARR (SEQ ID NO:38), YARAAARQARA (SEQ ID NO:39), YARAARRAARR (SEQ ID NO:40), and YARAARRAARA (SEQ ID NO:41), (See Ho et al. Cancer Res (2001) 61:474-477), R₉-Tat (GRRRRRRRRRPPQ; SEQ ID NO:2), HIV-I Rev-(34-50) (TRQARRNRRRRWRERQR; SEQ ID NO:4), FHV Coat (35-49) (RRRRNRTRRNRRRVR; SEQ ID NO:5), BMV Gag-(7- 25) (KMTRAQRRAAARRNRWTAR; SEQ ID NO:6), HTLV-II Rex-(4-16) (TRRQRTRRQRRNR; SEQ ID NO:7), CCMV Gag-(7-25) (KLTRAQRRAAARKNKRNTR; SEQ ID NO:8), P22 N-(14-30)

NAKTRRHERRRKLAIER; SEQ ID NO: 9), W/R (RRWRRWWRRWWRRWRR; SEQ ID NO: 10), NLS (TPPKKKRKVEDP; SEQ ID NO:11), AIkCWK₁₈ (CWKKKKKKKKKKKKKKKKKK; SEQ ID NO: 12), K₁₆RGD (SEQ ID NO: 15), Pl (K_I6GGCMFGCGG; SEQ ID NO:16), P2 (K_I6ICRRARGDNPDDRCT; SEQ ID NO:17), MPG (GALFLGFLGGAAGSTMGAWSQPKSKRKV; SEQ ID NO:22, a chimera of part of the HIV gp41 protein and the NLS of the SV40 large T antigen), (LAR)_2-I0 (SEQ ID NO:42), residues 43-58 of the third helix of the Drosophila antennapedia homeodomain (AntP or Ant PTD, also known as penetratin; RQIKIWFQNRRMKWKK; SEQ ID NO:3), PTD-4 (PIRRRKKLRRLK; SEQ ID NO:43), PTD-5 (RRQRRTSKLMKR; SEQ ID NO:44), KRIHPRLTRSIR (SEQ ID NO:45), PPRLRKRRQLNM (SEQ ID NO:46) and KLALKLALKALKAALKLA (SEQ ID NO:47). Preferably, the cationic peptide is selected from the group consisting of any one of AntP (RQIKIWFQNRRMKWKK; SEQ ID NO:3), HIV Tat (GRKKRRQRRRPPQ; SEQ ID NO:29) or HIV Tat dimer (CGRKKRRQRRRPPQGRKKRRQRRRPPQ; SEQ ID NO:30) or a combination thereof.

One skilled in the art can readily determine the ability of additional cationic peptide to mediate RNA transfection using methods known to those skilled in the art. Preferably, protein production of one or more polypeptides encoded by the transfected RNA is measured. Methods for detecting proteins are known to those skilled in the art, and include, but are not limited to Western blotting, extracellular staining, detection of markers such as antibiotic resistance, luciferase, /3-galactosidase, and the like. Alternatively, labeled RNA (e.g., fluorescently labeled or ³²P labeled RNA) is premixed with a cationic peptide of interest and then added to a cell culture, incubated for approximately thirty to sixty minutes, the culture media is removed and the cells are washed, and then label remaining in the cells is measured using standard techniques.

The peptides of the invention may be used to target RNA to the cytoplasm or to a specific organelle of a cell, such as the nucleus. Preferably the RNA is targeted to the cytoplasm. Many prior art translocatable peptides, such as the NLS of the SV40 large T antigen, MPG and loligomer consist of, or include, nuclear localization signals. In contrast, preferred cationic peptides of the invention do not contain a nuclear localization signal.

In a preferred embodiment, the RNA is a translatable RNA and the cationic peptides and the cationic peptide-RNA complexes of the invention preferentially target, or are localized in the cytoplasm. Cytoplasmic targeting is preferred because this is where RNA translation occurs. In one embodiment, the cationic peptide consists essentially of a peptide transduction domain.

Protein translated from RNA delivered to a cell by the methods of the invention can be detected by methods known in the art. A variety of techniques are available in the art for protein analysis and include, but are not limited to radioimmunoassays, ELISA (enzyme linked imrnunoradiometric assays), "sandwich" immunoassays, immunoradiornetric assays, in situ immunoassays (using e.g., colloidal gold, enzyme or radioisotope labels), western blot analysis, immunoprecipitation assays, immunofluorescent assays and PAGE-SDS.

For the purposes of the invention, a peptide transduction domain refers to an amino acid sequence that preferentially localizes in the cytoplasm, as opposed to other cell organelles (e.g., membrane, nucleus, mitochondria, plastid endoplasmic reticulum, golgi apparatus and the like). Preferably, at least 50%, 60%, 70%, 80%, or at least 90% of the cationic peptides of the invention are localized to the cytoplasm. The intracellular location(s) of a peptide can easily be determined by fluorescently tagging the peptide, and by a variety of other routine methods known to those of skill in the art.

In a preferred embodiment, the cationic peptide of the invention consists of, consists essentially of or comprises a T cell helper epitope (MHC helper epitope). For the purposes of the invention, T cell helper epitope, helper epitope, MHC helper epitope and T cell epitope are used interchangeably and refer to a peptide that specifically binds to a MHC Class I or MHC Class II molecule. Methods of detecting and measuring binding of peptide to MHC molecules are known to those skilled in the art. See, for example, U.S. 5,747,269, the contents of which are incorporated by reference. T cell helper epitopes are useful for enhancing the response of T cells to antigen presenting cells, such as dendritic cells. Thus, use of a cationic peptide that contains a T cell helper epitope is preferred for RNA transfection of antigen presenting cells.

The terms "major histocompatibility complex" or "MHC" refers to a complex of genes encoding cell-surface molecules that are required for antigen presentation to T cells. In humans, the MHC is also known as the "human leukocyte antigen" or "HLA" complex. The proteins encoded by the MHC are known as "MHC molecules" and are classified into class I and class II MHC molecules. CD8⁺ T cells respond to antigen presented by MHC Class I molecules, while CD4⁺ T cells (also known as T helper cells) respond to antigen presented by MHC Class II molecules. Human MHC Class I molecules include HLA-A, -B, and -C in humans. Human MHC Class II molecules include HLA-DP, -DQ, and -DR. In a preferred embodiment, the T cell helper epitope can complex with MHC molecules of any HLA type. Those of skill in the art are familiar with the serotypes and genotypes of the HLA. See: http://bimas.dcrt.nih.gov/cgi-bin/molbio/hla_coefficient_viewing_page, Rammensee et al. MHC Ligands and Peptide Motifs (1997) Chapman & Hall Publishers; and Schreuder et al. The HLA dictionary (1999) Tissue Antigens 54:409-437. The ability of a peptide to bind an MHC Class I molecule can be predicted using software available at thr.cit.nih.gov/molbio/hla_bind/, an http web site. The world wide web site, syfpeithi.de/, can be also be used to predict which peptides bind to MHC class I and class II molecules.

A T cell helper epitope binds to a MHC molecule to form a complex, which is displayed on the cell surface. For the purposes of the invention, "MHC Class I helper epitope" refers to a T cell helper epitope that binds to a MHC Class I molecule, while "MHC Class II helper epitope" refers to a T cell helper epitope that binds to a MHC Class II molecule. The MHC Class I molecule: MHC Class I helper epitope complex can enhance the interaction of antigen presenting cells with CD8⁺ T cells, while the MHC Class II molecule: MHC Class II helper epitope complex can enhance the interaction of antigen presenting cells with CD4⁺ T cells. Inclusion of a T cell helper epitope (binding peptide) can thereby facilitate the interaction of antigen presenting cells with T cells.

Binding motifs of T cell helper epitopes that bind to MHC Class I molecules and motifs of helper epitopes that bind to MHC Class II molecules are known to those skilled in the art. See the JenPep Database (ienner.ac.uk/ienpep at the world wide web), which currently contains a compendium of over 3200 T cell helper epitope sequences; Guttinger et al. 1988 EMBO J 7:2555-2558; Tabatabai et al. 199-9 Hum Immunol 60:105-115; Dadaglio et al. 1991 J Immunol 147:2302-2309, Nisini et al. 1997 J Virol 71:2241-2251, the contents of which are incorporated by reference. In addition, algorithms for predicting MHC class I and MHC class II helper peptides are known to those skilled in the art. See, for example, Southwood et al. 1998 J Immunol. 160:3363-3373, the contents of which are incorporated by reference. MHC class I helper epitopes are typically 8-10 residues in length, while MHC class II helper epitopes are typically 10 to 20 residues in length, and most often 13 to 16 residues in length. Examples of MHC class I helper epitopes are disclosed in Sidney et al. 1996 Hum. Immunol. 45:79; Sidney et al. 1996 Immunol. Today 17:261; Sidney et al. 1995 J. Immunol. 154:247; del Guerico et al. 1995 J. Immunol. 154:685; Sidney et al. 1996 J. Immunol. 157:3480 and Set et al. 2003 Immunogenetics 54:830-841, the contents of which are incorporated by reference. Examples of 9-mer cores of T cell helper epitopes that bind to MHC class II molecules include, but are not limited to, MSTPEATGM (SEQ ID NO:48), ISTAPVQMP (SEQ ID NO:49), VSTQLIMPG (SEQ ID NO:50), LVLMAVVLA (SEQ ID NO:51), MPTAESTGM (SEQ ID NO:52), MLGTHTMEV (SEQ ID NO:53), LIGANASFS (SEQ ID NO:54), LQAAIPLTS (SEQ ID NO:55), VTAQWLQA (SEQ ID NO:56) and LRNQPLTFA (SEQ ID NO:57) (See Kobayashi et al. 2001 Can. Res. 61:7577-7584, the contents of which are incorporated by reference). One or more cationic residues may be added to the amino and/or carboxy terminus of a T cell helper epitope to form a cationic peptide with an embedded helper epitope.

Assays to measure MHC binding to MHC class I helper epitopes, as well as MHC class II helper epitopes are disclosed in Sidney et al. 2002 J. Immunol. 169:5098-5108, the contents of which are incorporated by reference. For example, dendritic cells loaded with a test MHC class II helper epitope can be contacted with autologous CD4+ T cells, and CD4+ T cell activation can then be measured by a variety of methods known to those of skill in the art. Particularly useful helper epitopes will induce proliferation of CD4+ T cells and secretion of ThI cytokines, such an INFγ and IL-2.

The T cell helper epitope may be fused to the cationic peptide, with or without intervening amino acids. In a preferred embodiment, the helper epitope is embedded within the cationic peptide. The term "embedded" when applied to a cationic peptide of the invention is used to refer to sequences wherein amino acid residues associated with a minimal helper epitope also participate in the binding or transduction of KNA. Embedding the helper epitope in the cationic peptide reduces the costs of peptide synthesis, and avoids the use of longer cationic peptides which may be toxic to the target cell. The cationic peptides of the invention with embedded T cell helper epitopes are preferably not more than 30 amino acids in length, preferably they are between 9-24 amino acids in length, most preferably 10-22 amino acids in length. In preferred embodiments, the cationic peptide contains a helper epitope flanked by one or more cationic amino acid residues. In one embodiment of the invention, cationic amino acid residues are added to either the amino or the carboxy terminus. Preferably, cationic amino acid residues are added to both the amino and carboxy terminus of a T helper epitope. The flanking cationic amino acid residues can also be interspersed with non-cationic amino acid residues. In a preferred embodiment, the helper epitope is flanked by one to three cationic amino acid residues, most preferably by one to three lysine residues. One preferred cationic peptide with an embedded tetanus toxoid helper epitope is RRKAOYIKANSKFIGITELKRH (SEQ ID NO:31). Other preferred cationic peptides containing and embedded helper epitope are KKKHIEKYLKKIKNSKKK (SEQ ID ΝO:32; a HLA-DR5 class II helper epitope with flanking cationic lysine residues), KKKVIKGGRHLIFCHSKKK.CDKKK (SEQ ID NO:33; a HLA-DRl 5 class II helper epitope with flanking cationic lysine residues), KKKPKYVRONTLKLATKKK (SEQ ID ΝO:34; a HLA-DRl class II helper epitope with flanking cationic lysine residues) and KKKKALENKKKOLGAGGKNKKK. (SEQ ID ΝO:35; a class II helper epitope with specificity for HLA-DRl 1 subtype DRBl 1101 and DRBl 1102 alleles, and HLA-DR14 DRBl 1401 alleles, and flanking cationic lysine residues). In the foregoing examples, the helper epitope is underlined, and the helper consensus sequence is shown in italics. In one embodiment of the invention, the helper epitope has a net positive charge at pH 7.0 and therefore is a cationic peptide itself, so that the addition of flanking cationic amino acid residues is not necessary for efficient transfection. The peptides of the invention can be obtained by a variety of methods known to those skilled in the art, such as chemical synthesis using a commercially available automated peptide synthesizer such as those manufactured by Perkin Elmer/ Applied Biosystems, Inc., Model 430A or 43 IA, Foster City, CA, USA. The synthesized protein or polypeptide can be precipitated and further purified, for example by high performance liquid chromatography (HPLC). Preferably, solid phase peptide synthesis is done using an automated peptide synthesizer such as, but not limited to, an Applied Biosystems Inc. (ABI) model 43 IA using the "Fastmoc" synthesis protocol supplied by ABI.

Alternatively, the peptides can be obtained by methods known to those skilled in the art, such as, but not limited to, expression in a biological system including bacterial, mammalian, insect, plant and viral systems (Maniatis, T. Molecular Cloning, A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1990).

Methods of designing and screening additional cationic peptides, such as degenerate oligonucleotide libraries for translocation, transfection and targeting ability are known to those skilled in the art. See, for example, U.S. Patent Application No. 20020172979, the content of which is incorporated by reference.

RNAs

The cationic peptides of the invention can be used to transform any type of cell with any type of RNA, including, but not limited to primary RNA transcripts, mRNA, rRNA, tRNA, dsRNA, siRNA (RNAi), ribozymes, antisense RNA, amplified RNA and the like. In a preferred embodiment, the RNA is a translatable RNA. By translatable RNA is meant that the RNA contains functional translation initiation and termination signals that flank an open reading frame, and that it is translated into a peptide or polypeptide upon introduction into a cell with translational machinery that recognizes the translation initiation and termination signals.

RNA can be extracted from one or more cells or produced in vitro using conventional molecular techniques. RNA may be amplified by reverse transcription to cDNA, followed by PCR amplification and in vitro transcription. Such methods are known to those skilled in the art. See, for example PCR Protcols, Eds. Innis et ai, Academic Press, Inc, Sand Diego, CA, 1990. Preferably, RNA is prepared according the methods of Heiser et al. (J. Immunol., 2001, 166:2953-2960) or as described in U.S. provisional application 60/525,076 filed 25 November 2003, the contents of which are incorporated by reference. Preferably, the RNA is a translatable RNA. Translatable RNA will contain a ribosome binding site and start codon. Preferably, the mRNA will also contain a 5' cap, stop codon and polyA tail.

In a preferred embodiment, the RNA is from a pathogen or a neoplastic (tumor) cell, such as a cancer cell. In preferred embodiments, the antigen is from a tumor cell or a pathogen. Preferably, the tumor cell is a renal cancer carcinoma cell, a multiple myeloma cell, a chronic lymphocytic leukemia cell or a melanoma cell. Preferred pathogens are HTV and HCV. In preferred embodiments, the antigen is delivered to the antigen presenting cell in the form of RNA isolated or derived from a cancer cell or a pathogen. Methods for RT-PCR of RNA extracted from any cell (e.g., a cancer cell or pathogen cell), and in vitro transcription are disclosed in copending U.S. provisional patent application No. 60/525,076, the contents of which are incorporated by reference. Pathogen nucleic acids can be obtained from a pathogen cell,

If desired, cell-specific RNA can be isolated by subtractive hybridization. For example, tumor-specific RNA could be isolated by extraction of RNA from tumor cells and subtractive hybridization with cDNA from non-tumor cells. Such methods are known to those of skill in the art.

Target cells

The methods and compositions of the invention may be used to transform any type of cell, including prokaryotic and eukaryotic cells, cells in culture, cells in tissue slices, or cells in an animal, including humans. In a preferred embodiment, the cell is a eukaryotic cell. In one embodiment of the inventions, the target cell is an antigen presenting cell. Antigen presenting cells include, but are not limited to, macrophages, including alveolar macrophages, peritoneal macrophages, and splenic macrophages, monocytes, dendritic cells, including Langerhans cells, immature dendritic cells and mature dendritic cells.

In a preferred embodiment, the target cell is a dendritic cell. The term "dendritic cells (DC)" refers to a diverse population of morphologically similar cell types found in a variety of lymphoid and non-lymphoid tissues (Steinman (1991) Ann. Rev. Immunol. 9:271-296). Dendritic cells are the most potent of the APCs, and provide the signals necessary for T cell activation and proliferation. Dendritic cells are derived from bone marrow progenitor cells, circulate in small numbers in the peripheral blood and appear either as immature Langerhans' cells or terminally differentiated mature cells. Dendritic cells can also be differentiated from monocytes. Methods for the isolation of antigen presenting cells (APCs), and for producing dendritic cell precursors and mature dendritic cells are known to those skilled. See, for example, U.S. Patent Applications 20030199673 and 20020164346, and WO 93/20185 the contents of which are incorporated by reference.

In a preferred embodiment, the target cell is an immature dendritic cell. Immature DC cells can be isolated or prepared from a suitable tissue source containing DC precursor cells and differentiated in vitro to produce immature DC. For example, a suitable tissue source can be one or more of bone marrow cells, peripheral blood progenitor cells (PBPCs), peripheral blood stem cells (PBSCs), and cord blood cells. Preferably, the tissue source is a peripheral blood mononuclear cell (PBMC). The tissue source can be fresh or frozen. In another aspect, the cells or tissue source are pre-treated with an effective amount of a growth factor that promotes growth and differentiation of non-stem or progenitor cells, which are then more easily separated from the cells of interest. These methods are known in the art and described briefly in Romani, et al. (1994) J. Exp. Med. 180:83 and Caux, C. et al. (1996) J. Exp. Med. 184:695.

Stem cells can be differentiated into dendritic cells by incubating the cells with the appropriate cytokines. Inaba et al. (1994) supra, described the in vitro differentiation of murine stem cells into dendritic cells by incubating the stem cells with murine GM-CSF. In brief, isolated stem cells are incubated with between 1 and 200 ng/ml murine GM-CSF, and preferably about 20 ng/ml GM-CSF in standard RPMI growth medium. The media is changed with fresh media about once every other day. After 7 days in culture, a large percentage of cells are dendritic, as assessed by expression of surface markers and morphology. Dendritic cells are isolated by florescence activated cell sorting (FACS) or by other standard methods.

Immature dendritic cells can be prepared from CD34⁺ hematopoietic stem or progenitor cells. The CD34⁺ hematopoietic stem or progenitor cells can be isolated from a tissue source selected from the group consisting of bone marrow cells, peripheral blood progenitor cells (PBPCs), peripheral blood stem cells (PBSCs), and cord blood cells. Human cells CD34 ⁺ hematopoietic stem cells are preferably differentiated in vitro by culturing the cells with human GM-CSF and TNF-α. See for example, Szabolcs, et al. (1995) 154:5851- 5861.

For mouse DCs, murine stem cells can be differentiated into dendritic cells by incubating the stem cells in culture with murine GM-CSF. Typically, the concentration of GM-CSF in culture is at least about 0.2 ng/ml, and preferably at least about 1 ng/ml. Often the range will be between about 20 ng/ml and 200 ng/ml. In many preferred embodiments, the dose will be about 100 ng/ml. IL-4 is optionally added in similar ranges for making murine DCs.

When human cells are transduced, human GM-CSF is used in similar ranges, and TNF-α also is added to facilitate differentiation. TNF-α is also typically added in about the same ranges. Optionally, SCF or other proliferation ligand (e.g., Flt3) is added in similar dose ranges to make human DCs.

Preferably, the immature DCs are prepared from peripheral blood mononuclear cells (PBMCs). In a preferred embodiment, the PBMCs are treated with an effective amount of granulocyte macrophage colony stimulating factor (GM-CSF) in the presence or absence of interleukin 4 (IL-4) and/or IL-13, so that the PBMCs differentiate into immature DCs. Most preferably, PBMCs are cultured in the presence of GM-CSF and IL-4 to produce immature DCs, suitable for use in the methods of the invention.

Immature dendritic cells may be matured into mature dendritic cells by methods known to those of skill in the art. See, for example, copending U.S. provisional application 60/522,512, filed 7 October 2004, the contents of which is incorporated by reference. Immature or mature dendritic cells may be transfected using cationic peptide:RNA complexes of the invention. Transfected dendritic cells are useful in the treatment of diseases, such as tumors and pathogen infections.

As is apparent to those of skill in the art, all of the above-noted dose ranges for differentiating stem cells are approximate. Different suppliers and different lots of cytokine from the same supplier vary in the activity of the cytokine. One of skill can easily titrate each cytokine which is used to determine the optimal dose for any particular cytokine.

Cationic Peptide-RNA Complexes

The invention further provides a composition comprising an isolated noncovalent complex of a cationic peptide and a RNA. The positively charged cationic peptide and the negatively charged RNA form a non-covalent complex through ionic bonding. The cationic peptide-RNA complexes of the invention encompass complexes that form between a single cationic peptide and multiple RNAs, between multiple cationic peptides and a single RNA, or between a single cationic peptide and a single RNA. The length, sequence and charge of the cationic peptide, as well as the length and sequence of the RNA, and the concentrations of each will be relevant factors in determining the ratio of cationic peptide:RNA. One skilled in the art can easily optimize the ratio and concentrations of cationic peptide and RNA for efficient complex formation and transfection. Preferred ratios are between 1-100 cationic peptides per RNA molecule.

The cationic peptide-RNA compositions of the invention shall not contain an amount of cationic lipid, neutral lipid, dendrimers, chloroquine, lysosomotropic agent, or other agents sufficient to increase, by 5% or more, the transfection efficiency of the cationic peptide-RNA complex in comparison to the transfection efficiency of the cationic peptide-RNA complex in the absence of such contaminants).

The cationic peptide-RNA complex can be formed prior to adding the cationic peptide and RNA to an environment containing a target cells for transfection, or the complex can be formed in the environment containing the target cells for transfection. For example, if the target cells are cells in tissue culture, a preformed cationic peptide-RNA complex can be added to the tissue culture medium, or RNA and cationic peptide can be added separately and allowed to associate in the culture medium. If the target cells are in a higher organism, it is preferred that a preformed cationic peptide-RNA is introduced into the organism. In one embodiment, cationic peptide and RNA are mixed in PBS and incubated at room temperature for 1-60 minutes, or overnight to allow peptide :RNA complexes to form. The temperature of incubation is not critical, and in preferred embodiments ranges between 1-5O⁰C.

The invention also provides pharmaceutical compositions comprising the cationic peptide-RNA complexes and a pharmaceutically acceptable carrier. Methods for formulating such compositions are known to those skilled in the art. Preferably, the cationic peptide- RNA complexes are in a physiologically acceptable solution, such as, but not limited to, sterile saline or sterile buffered saline. Pharmaceutically acceptable carriers or buffer solutions are known in the art and include those described in a variety of texts such as Remington's Pharmaceutical Sciences.

Transformation of Target Cells

The cationic peptide and nucleic acid can be delivered to cells in vivo or ex vivo. The cationic peptide and nucleic acid can be used at any RNA concentration effective to result in uptake of the RNA or cationic peptide:RNA complex into cells. Such effective concentrations typically range from 1 ng/ml to 1 mg/ml RNA in cell culture. Optimal concentrations for in vitro and in vivo use can be determined by those skilled in the art.

In a preferred embodiment, the cationic peptide and RNA are mixed in an aqueous solution and then added together to a cell culture medium or administered to an animal, preferably to a human. Preferably the cationic peptide and RNA are incubated for a time sufficient to allow complex formation prior to adding the solution to a cell culture medium or to administration. Alternatively, the cationic peptide and RNA can be added separately, and in either order, to an environment containing a target cell, such as tissue culture or a human or animal subject.

For transfection of cells in tissue culture, the cells can be washed with a physiological buffer, such as PBS, prior to adding the cationic peptide and RNA. The cells can be contacted with the cationic peptide and RNA (preferably as a preformed complex) for any length of time. Preferably, the cells are contacted with the cationic peptide and RNA for at least one minute, and preferably 5 minutes to 2 hours. If a transfection medium is used, the transfection medium may be removed after the contact period and replaced with an appropriate cell culture medium.

The invention further provides a vaccine comprising the loaded antigen presenting cells are described above. In such vaccines, the loaded antigen presenting cells will be in a buffer suitable for therapeutic administration to a patient. The vaccine may further comprise an adjuvant for factors for the stimulation of antigen presenting cells or T cells. Methods of formulating pharmaceutical compositions are known to those skilled in the art. See, for example, the latest version of Remington's Pharmaceutical Science.

The optimal immunization interval for dendritic cell vaccines can be determined by one of skill in the art. In a preferred embodiment, patients will be vaccinated 5 times with between IxIO⁶ to IxIO⁷ viable RNA-loaded DCs per dose. The dose level selected for vaccination is expected to be safe and well-tolerated.

Methods of isolating, preparing, transfecting, formulating and administering antigen presenting cells to patients is known in the art. See, for example, Fay et al. Blood 2000 96:3487; Fong et al. J Immunol 2001b 166:4254-4259; Ribas et al. Proc Am Soc Clin One 2001 20:1069; Schuler-Thurneret al. J Exp Med. 2002 195:1279-88. Erratum in: J Exp Med. 2003197:395; and Stift et al. J Clin Oncol 2003 21: 135-142, the contents of which are incorporated by reference.

In vivo methods of delivery include, but are not limited to, intravenous, intramuscular, oral, nasal, topical, mucosal, intradermal, intrathecal, intraperitoneal, subcutaneous, cutaneous and osmotic delivery. For in vivo delivery, the complex may be combined with a pharmaceutically acceptable carrier. Such carriers are known to those skilled in the art. See, for example, Remington's Pharmaceutical Sciences, most recent edition, E. W. Martin (Ed.) Mack Publishing Co., Easton, PA. Typical routes of APC administration employed clinically include, but are not limited to, intravenous (IV), subcutaneous (SC), intradermal (ID), and intralymphatic. Objective clinical responses have been reported following IV, SC, and ID dosing. Currently, there is a developing preference for ID administration since the dermis is a normal residence for dendritic cells from which they are known to migrate to draining lymph nodes. In murine models, SC-injected dendritic cells are later found in T cell areas of draining lymph nodes and trigger protective antitumor immunity that is superior to that following IV immunization. There is murine evidence that dendritic cell injection directly into a lymph node is superior to other routes of delivery in generating protective antitumor immunity or cytotoxic T-lymphocytes (CTLs) (Lambert et al. Cancer Res 2001 61:641-646, the contents of which are incorporated by reference). This suggests that an entire dendritic cell dose should be delivered so that it impacts on a single draining lymph node or basin (rather than dividing the dose among multiple sites to engage as many nodes as possible). To assess the immunogenicity of the vaccine, immune responses in vaccinated individuals can be monitored by following the maturation profiles of CD4+ and CD8+ T cells.

The methods of the invention specifically exclude the use of polyethylenimine, dendrimers, chloroquine or other lysosomotropic agents, or exogenous cationic lipids in an amount or concentration effective to mediate transfection. Such cationic lipids include DOTAP, DOTMA, DOPE, DOSPA, DODAC, CHOL, DMEDA, DDAB, DODAC, DORI, DORIE, DOSPA, DOGS, DPPES, DOSPER, Lipofectamine™, Lipofectin™, Lipofectace™, and the like. However, the method of the invention do not exclude cationic lipids that may be normally present in cell cultures, nucleic acid extracts, in vitro transcription reactions, and the like, and in an animal or human subject. Dendrimers are a type of synthetic polymer with regular dendric branching with radial symmetry composed of an initiator core and repeating units radially attached to the core and an exterior surface of functional groups. See for example, U.S. Pat no. 5,527,524; 5,338,532; 4,693,064; 4,568,737 and 4,507,466. The practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. These methods are described in the following publications. See, e.g., Sambrook et al. MOLECULAR CLONING: A LABORATORY MANUAL, 2nd edition (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F. M. Ausubel et al. eds. (1987)); the series METHODS IN ENZYMOLOGY (Academic Press, Inc.); PCR: A PRACTICAL APPROACH (M. MacPherson et al. IRL Press at Oxford University Press (1991)); PCR 2: A PRACTICAL APPROACH (M. J. MacPherson, B. D. Hames and G. R. Taylor eds. (1995)); ANTIBODIES, A LABORATORY MANUAL (Harlow and Lane eds. (1988)); and ANIMAL CELL CULTURE (R. I. Freshney Ed. (1987)).

The following examples are intended to illustrate, but not limit the invention.

EXAMPLES

Example 1 Cationic Peptide-Mediated Transfection of GFP RNA into HeLa cells

The Port-2 (AntP: RQIKIWFQNRRMKWKK; SEQ ID N0:3), Port-3 (HIV Tat: GRKKRRQRRRPPQ; SEQ ID NO:29) or Port-4 (Port-3 dimer:

CGRKKRRQRRRPPQGRKKRRQRRRPPQ; SEQ ID NO:30) cationic peptides are used to transfect RNA encoding green fluorescent protein (GFP) into HeLa cells. Port cationic peptide:RNA complex ratios range from 100:1 to 1:1. The Port cationic peptide:RNA rations are calculated for both the ration of peptide molecules to RNA molecules and by the charge ratio between the molecules. Port peptide is diluted in PBS at concentrations appropriate for the desired ration. 100 μ\ of Port peptide is mixed with 100 μl RNA (20 /xg/ml) and incubated at room temperature for 30 minutes to allow complexes to form. Adherent HeLa cells are washed with 4 ml PBS. The 200 μ\ Port:RNA complex is added drop wise to washed cells, followed by 400 μ\ serum free media (DMEM). Cells overlaid with Port-RNA complex solution are incubated for one hour at 37⁰C, and then 1 ml complete HeLa medium (DMEM supplemented with 10% FBS) is added to cells. The cells are then incubated at 5 022705

37°C overnight, harvested by trypsin digestion and assayed for GFP expression by FLOW cytometry.

Example 2 DC generation and transfection

Human PBMCs are isolated from Leukapheresis collections. PBMCs are prepared by Ficoll®-histopaque density centrifugation and washed four times in PBS at room temperature. 2x 10⁸ PBMCs are re-suspended in 30ml AIM-V medium and allowed to adhere to 150 cm³ plastic flasks for 2 hours at 37⁰C. Non-adherent cells are removed and remaining cells cultured in X- vivo 15 medium, supplemented with GM-CSF (800 U/ml) and IL-4 (500U/ml), for 6 days at 37°C, 5% CO₂. Immature DC can then be transfected by contact with the cationic peptide RNA complexes of the invention. 2-20 μg in vitro translated mRNA is incubated with a 5 molar excess of the cationic peptide of SEQ ID NO:31 in PBS for one hour, and added to a flask of immature dendritic cells and incubated at 37°C, 5% CO₂ for 2 hours. Transfected immature DC may be administered to a subject in the immature or first matured as described below.

Example 3 DC maturation

Immature DCs are matured with a "cytokine cocktail" comprising of TNF-α (10 ng/ml), IL-1/3 (10 ng/ml), IL-6 (100 ng/ml) and PGE₂ (1 μg/ml) and incubated overnight at 37⁰C, 5% CO₂. Mature DC can be transfected by contact with the cationic peptide RNA complexes of the invention. Transfected mature DC can then be administered to a subject or cryopreserved for subsequent administration.

Throughout this disclosure, various publications, patents and published patent specifications are referenced by an identifying citation. The disclosures of these publications, patents and published patent specifications are hereby incorporated by reference into the present disclosure to more fully describe the state of the art to which this invention pertains.

Claims

05CLAIMS

1. A method of transforming a cell, comprising: contacting a cell with a cationic peptide and a RNA.

2. The method of claim 1 , wherein the cationic peptide and the RNA form a non- covalent complex.

3. The method of claim 2, wherein the non-covalent complex is formed prior to contacting the cell, by steps of (a) adding the cationic peptide to the RNA to form a mixture, and (b) incubating the mixture for a time sufficient to allow the formation of a non-covalent complex between the cationic peptide and the RNA.

4. The method of claim 1 , wherein the contacting is performed in vitro.

5. The method of claim 1, wherein the cationic peptide is an isolated peptide, a synthetic peptide, or a recombinant peptide.

6. The method of claim 1 , wherein said peptide is 8 to 22 amino acids in length.

7. The method of claim 1 , wherein said peptide is 10-21 amino acids in length.

8. The method of claim 1 , wherein said cationic peptide does not contain a nuclear localization signal.

9. The method of claim 1 , wherein said peptide consists essentially of a peptide transduction domain.

10. The method of claim 1 , wherein said peptide contains a single peptide transduction domain and does not contain a nuclear localization signal.

11. The method of claim 1 , wherein said complex is preferentially localized to the cytoplasm of said cell.

12. The method of claim 1 , wherein said cationic peptide is selected from the group consisting of AntP (RQIKIWFQNRRMKWKK; SEQ ID NO:3), HIV Tat (GRKKRRQRRRPPQ; SEQ ID NO:29) or HIV Tat dimer (CGRKECRRQRRRPPQGRKKRRQRRRPPQ; SEQ ID NO:30) or a combination thereof.

13. The method of claim 1 , wherein said peptide comprises a MHC helper epitope.

14. The method of claim 13, wherein said MHC helper epitope is a MHC II helper epitope.

15. The method of claim 13 , wherein a helper epitope is embedded in said cationic peptide.

16. The method of claim 15, wherein said cationic peptide is selected from the group consisting of RRKAQYIKANSKFIGITELKRH (SEQ ID NO:31), KKKHIEKYLKKIKNSKKK (SEQ ID NO:32), KKKVIKGGRHLIFCHSKKKCDKKK (SEQ ID NO:33), KKKPKYVRQNTLKLATKKK (SEQ ID NO:34),

KKKKALENKKKQLGAGGKNKKK (SEQ ID NO:35) and any combination thereof.

17. The method of claim 1, wherein said cationic peptide comprises one or more D- amino acid residues.

18. The method of claim 1 , wherein said RNA is selected from the group consisting of mRNA, antisense RNA, RNAi, amplified RNA and a ribozyme.

19. The method of claim 1, wherein said RNA is a translatable RNA.

20. The method of claim 1, wherein said RNA is isolated from one or more tumor cells or pathogen.

21. The method of claim 20, wherein said tumor cell is selected from renal cell carcinoma, melanoma or chronic lymphocytic leukemia.

22. The method of claim 20, wherein the pathogen is HIV or HCV.

23. The method of claim 18, wherein said RNA is amplified from RNA isolated from one or more tumor cells or pathogens.

24. The method of claim 1, wherein said cell is an antigen presenting cell.

25. The method of claim 24, wherein said antigen presenting cell is a dendritic cell.

26. The method of claim 25, wherein said dendritic cell is an immature dendritic cell.

27. The method of claim 25, wherein said dendritic cell is a mature dendritic cell.

28. The method of claim 1 , wherein said contacting is performed in the absence of a cationic lipid or a dendrimer.

29. A composition comprising an isolated noncovalent complex of a cationic peptide and an RNA.

30. A composition comprising an antigen presenting cell transformed with a noncovalent complex comprising a cationic peptide and a RNA.

31. The composition of claim 29 or 30, wherein said peptide is 8 to 22 amino acids in length.

32. The composition of claim 31, wherein said peptide is 10 to 20 amino acids in length.

33. The composition of claim 29 or 30, wherein said cationic peptide does not contain a nuclear localization signal.

34. The composition of claim 29 or 30, wherein said peptide consists essentially of a peptide transduction domain.

35. The composition of claim 29 or 30, wherein said peptide contains a single peptide transduction domain and does not contain a nuclear localization signal.

36. The composition of claim 29 or 30, wherein said complex is preferentially localized to the cytoplasm of said cell.

37. The composition of claim 29 or 30, wherein said cationic peptide is selected from the group consisting of AntP (RQIKIWFQNRRMKWKK; SEQ ID NO:3), HIV Tat (GRKKRRQRRRPPQ; SEQ ID NO:29), HIV Tat dimer (CGRKKRRQRRRPPQGRKKRRQRRRPPQ; SEQ ID NO:30) and any combination thereof.

38. The composition of claim 29 or 30, wherein said peptide comprises a MHC helper epitope.

39. The composition of claim 38, wherein said MHC helper epitope is a MHC II helper epitope.

40. The composition of claim 38, wherein a helper epitope is embedded in said cationic peptide.

41. The composition of claim 40, wherein said cationic peptide is selected from the group consisting of RRKAQYIKANSKFIGITELKRH (SEQ ID NO:31), KKKHIEKYLKKIKNSKKK (SEQ ID NO:32), KKKVIKGGRHLIFCHSKKKCDKKK (SEQ ID NO-.33), KKKPKYVRQNTLKLATKKK (SEQ ID NO:34), 05

KKKKALENKKKQLGAGGKNKKK (SEQ ID NO-.35) and any combination thereof.

42. The composition of claim 29 or 30, wherein said cationic peptide comprises one or more D-amino acid residues.

43. The composition of claim 29 or 30, wherein said RNA is selected from the group consisting of mRNA, antisense RNA, RNAi, amplified RNA and a ribozyme.

44. The composition of claim 29 or 30, wherein said RNA is a translatable RNA.

45. The composition of claim 29 or 30, wherein said RNA is isolated from one or more tumor cell or pathogen.

46. The composition of claim 45, wherein said tumor cell is selected from renal cell carcinoma, melanoma or chronic lymphocytic leukemia.

47. The method of claim 45, wherein said pathogen is HIV or HCV.

48. The composition of claim 45, wherein said RNA is amplified from RNA isolated from one or more tumor cells or pathogen.

49. The composition of claim 30, wherein said cell is an antigen presenting cell.

50. The composition of claim 49, wherein said antigen presenting cell is a dendritic cell.

51. The composition of claim 50, wherein said dendritic cell is an immature dendritic cell.

52. The composition of claim 50, wherein said dendritic cell is a mature dendritic cell.

53. A composition comprising a cationic peptide selected from the groups consisting of AntP (RQIKIWFQNRRMKWKK; SEQ ID NO:3), HIV Tat (GRKKJRRQRRRPPQ; SEQ ID NO:29), HIV Tat dimer (CGRKKRRQRRRPPQGRKKRRQRRRPPQ; SEQ ID NO:30), RRKAQYIKANSKFIGITELKRH (SEQ ID NO:31) and any combination thereof.

54. A composition comprising a cationic peptide-RNA complex and a pharmaceutical acceptable carrier, wherein said cationic peptide and said RNA are noncovalently complexed.